Since its introduction in the early 1990's, optical coherence tomography (OCT) has emerged as a promising imaging modality for micrometer-scale noninvasive imaging in biological and biomedical applications. Its relatively low cost and real-time, in vivo capabilities have fueled the investigation of this technique for applications in retinal and anterior segment imaging in opthalmology (e.g., to detect retinal pathologies), early cancer detection and staging in the skin, gastrointestinal, and genitourinary tracts, as well as for ultra-high resolution imaging of entire animals in embryology and developmental biology.
Conventional OCT systems are essentially range-gated low-coherence interferometers that have been configured for characterization of the scattering properties of biological and other samples. By measuring singly backscattered light as a function of depth, OCT fills a valuable niche in imaging of tissue ultrastructure, and provides subsurface imaging with high spatial resolution (˜1-10 μm) in three dimensions and high sensitivity (>110 dB) in vivo with no contact needed between the probe and the tissue. OCT is based on the one-dimensional technique of optical coherence domain reflectometry (OCDR), also called optical low-coherence reflectometry (OLCR). See Youngquist, R. C., S. Carr, and D. E. N. Davies, Optical Coherence Domain Reflectometry: A New Optical Evaluation Technique. Opt. Lett., 1987. 12: p. 158; Takada, K., et al., New measurement system for fault location in optical wave guide devices based on an interferometric technique. Applied Optics, 1987. 26(9): p. 1603-1606; and Danielson, B. L. and C. D. Whittenberg, Guided-wave Reflectometry with Micrometer Resolution. Applied Optics, 1987. 26(14): p. 2836-2842. In some instances of time-domain OCT, depth in the sample is gated by low coherence interferometry. The sample is placed in the sample arm of a Michelson interferometer, and a scanning optical delay line is located in the reference arm.
The time-domain approach used in conventional OCT has been used in supporting biological and medical applications. An alternate approach involves acquiring as a function of optical wavenumber the interferometric signal generated by mixing sample light with reference light at a fixed group delay. Two methods have been developed which employ this Fourier domain (FD) approach. The first is generally referred to as Spectral-domain OCT (SD-OCT). SD-OCT uses a broadband light source and achieves spectral discrimination with a dispersive spectrometer in the detector arm. The second is generally referred to as swept-source OCT (SS-OCT). SS-OCT time-encodes wavenumber by rapidly tuning a narrowband source through a broad optical bandwidth. Both of these techniques can provide improvements in SNR of up to 15-20 dB when compared to time-domain OCT, because SD-OCT and SS-OCT capture the complex reflectivity profile (the magnitude of which is generally referred to as the “A-scan” data or depth-resolved sample reflectivity profile) in parallel. This is in contrast to time-domain OCT, where destructive interference is employed to isolate the interferometric signal from only one depth at a time as the reference delay is scanned.
However, the resolution of current OCT techniques is generally limited by the coherence length of the illumination source. Therefore, current OCT techniques may not be able to resolve structures of less than ˜1-10 μm. For example, the characteristics and dynamics of the cellular surface may be of interest in many areas of quantitative biology. However, there are few scientific tools which are capable of noninvasively acquiring quantitative information about cell surface profiles, displacements, and motions on the nanometer scale.
Recent advances have increased the imaging speed, which may allow relatively large image sets (such as 3-D volumes) to be quickly generated. Since OCT is high-speed, non-contact and non-destructive, it is well suited for imaging dynamics over short time scales, for example, well below 1 second (the beating of a heart tube in a fruit fly) all the way up to changes over a long time scales, for example, days or even longer (tissue growing).
Spectral domain phase microscopy (SDPM) is an extension of SD-OCT (38-40) which may allow for the measurement of nanometer-scale displacements and motions within each pixel of an SD-OCT image. The major modifications in SDPM as compared to SD-OCT are the substitution of a common path interferometer for the Michelson interferometer in SD-OCT, and enhanced signal processing based on the measured phase of the SD-OCT signal in each image pixel to extract nanometer-scale displacement and motion of reflectors and scatterers located within that pixel. SDPM has been discussed in, for example, “Spectral-domain phase microscopy” by M. A. Choma, A. K. Ellerbee, C. Yang, T. L. Creazzo, and J. A. Izatt, Opt. Lett. 30(10), 1162 (2005) and “Spectral-domain optical coherence phase microscopy for quantitative phase-contrast imaging” by C. Joo, T. Akkin, B. Cense, B. H. Park, and J. F. DeBoer, Opt. Lett. 30(16), 2131 (2005).